- Volume 150, Issue 7, 2004

The symbioses between cellulose-degrading flagellates and bacteria are one of the most fascinating phenomena in the complex micro-ecosystem found in the hindgut of lower termites. However, little is known about the identity of the symbionts. One example is the epibiotic bacteria colonizing the surface of hypermastigote protists of the genus Staurojoenina. By using scanning electron microscopy, it was shown that the whole surface of Staurojoenina sp. from the termite Neotermes cubanus is densely covered with long rod-shaped bacteria of uniform size and morphology. PCR amplification of 16S rRNA genes from isolated protozoa and subsequent cloning yielded a uniform collection of clones with virtually identical sequences. Phylogenetic analysis placed them as a new lineage among the Bacteroidales, only distantly related to other uncultivated bacteria in the hindgut of other termites, including an epibiont of the flagellate Mixotricha paradoxa. The closest cultivated relative was Tannerella forsythensis (<85 % sequence identity). Fluorescence in situ hybridization with a newly designed clone-specific oligonucleotide probe confirmed that these sequences belong to the rod-shaped epibionts of Staurojoenina sp. Transmission electron microscopy confirmed the presence of a Gram-negative cell wall and revealed special attachment sites for the symbionts on the cell envelope of the flagellate host. Based on the isolated phylogenetic position and the specific association with the surface of Staurojoenina sp., we propose to classify this new taxon of Bacteroidales under the provisional name ‘Candidatus Vestibaculum illigatum’.

Nitrogen fixation is catalysed by some photosynthetic bacteria. This paper presents a phylogenetic comparison of a nitrogen fixation gene (nifH) with the aim of elucidating the processes underlying the evolutionary history of Rhodopseudomonas palustris. In the NifH phylogeny, strains of Rps. palustris were placed in close association with Rhodobacter spp. and other phototrophic purple non-sulfur bacteria belonging to the α-Proteobacteria, separated from its close relatives Bradyrhizobium japonicum and the phototrophic rhizobia (Bradyrhizobium spp. IRBG 2, IRBG 228, IRBG 230 and BTAi 1) as deduced from the 16S rRNA phylogeny. The close association of the strains of Rps. palustris with those of Rhodobacter and Rhodovulum, as well as Rhodospirillum rubrum, was supported by the mol% G+C of their nifH gene and by the signature sequences found in the sequence alignment. In contrast, comparison of a number of informational and operational genes common to Rps. palustris CGA009, B. japonicum USDA 110 and Rhodobacter sphaeroides 2.4.1 suggested that the genome of Rps. palustris is more related to that of B. japonicum than to the Rba. sphaeroides genome. These results strongly suggest that the nifH of Rps. palustris is highly related to those of the phototrophic purple non-sulfur bacteria included in this study, and might have come from an ancestral gene common to these phototrophic species through lateral gene transfer. Although this finding complicates the use of nifH to infer the phylogenetic relationships among the phototrophic bacteria in molecular diversity studies, it establishes a framework to resolve the origins and diversification of nitrogen fixation among the phototrophic bacteria in the α-Proteobacteria.

Inspection of the genomes of Streptomyces coelicolor A3(2) and Streptomyces avermitilis reveals that each contains 55 putative eukaryotic-type protein phosphatases (PPs), the largest number ever identified from any single prokaryotic organism. Unlike most other prokaryotic genomes that have only one or two superfamilies of eukaryotic-type PPs, the streptomycete genomes possess the eukaryotic-type PPs that belong to four superfamilies: 2 phosphoprotein phosphatases and 2 low-molecular-mass protein tyrosine phosphatases in each species, 49 Mg2+- or Mn2+-dependent protein phosphatases (PPMs) and 2 conventional protein tyrosine phosphatases (CPTPs) in S. coelicolor A3(2), and 48 PPMs and 3 CPTPs in S. avermitilis. Sixty-four percent of the PPs found in S. coelicolor A3(2) have orthologues in S. avermitilis, indicating that they originated from a common ancestor and might be involved in the regulation of more conserved metabolic activities. The genes of eukaryotic-type PP unique to each surveyed streptomycete genome are mainly located in two arms of the linear chromosomes and their evolution might be involved in gene acquisition or duplication to adapt to the extremely variable soil environments where these organisms live. In addition, 56 % of the PPs from S. coelicolor A3(2) and 65 % of the PPs from S. avermitilis possess at least one additional domain having a putative biological function. These include the domains involved in the detection of redox potential, the binding of cyclic nucleotides, mRNA, DNA and ATP, and the catalysis of phosphorylation reactions. Because they contained multiple functional domains, most of them were assigned functions other than PPs in previous annotations. Although few studies have been conducted on the physiological functions of the PPs in streptomycetes, the existence of large numbers of putative PPs in these two streptomycete genomes strongly suggests that eukaryotic-type PPs play important regulatory roles in primary or secondary metabolic pathways. The identification and analysis of such a large number of putative eukaryotic-type PPs from S. coelicolor A3(2) and S. avermitilis constitute a basis for further exploration of the signal transduction pathways mediated by these phosphatases in industrially important strains of streptomycetes.

Activated sludge plants designed to remove phosphorus microbiologically often perform unreliably. One suggestion is that the polyphosphate-accumulating organisms (PAO) are out-competed for substrates by another group of bacteria, the glycogen-accumulating organisms (GAO) in the anaerobic zones of these processes. This study used fluorescence in situ hybridization (FISH) and denaturing gradient gel electrophoresis (DGGE) to analyse the communities from laboratory-scale anaerobic : aerobic sequencing batch reactors. Members of the genus Sphingomonas in the α-Proteobacteria were present in large numbers in communities with poor phosphorus removal capacity where the biomass had a high glycogen content. Their ability to store poly-β-hydroxyalkanoates anaerobically, but not aerobically, and not accumulate polyphosphate aerobically is consistent with these organisms behaving as GAO there. No evidence was found to support an important role for the γ-Proteobacteria as possible GAO in these communities, although these bacterial populations have been considered in other studies to act as possible competitors for the PAO.

At the onset of sporulation in Bacillus subtilis, an asymmetric cell division gives rise to two unequal-sized compartments with distinct developmental fates. The smaller compartment, or prespore, becomes the spore, whilst the larger compartment, or mother cell, eventually lyses after contributing to spore maturation. The fate of each compartment is determined by differential gene expression, controlled by the activation of four compartment-specific σ-factors. The expression and activity of all four σ-factors are tightly regulated to ensure the correct sequence of morphological events. Prespore-specific genes are transcribed by two σ-factors, σ F followed by σ G. The gene encoding σ G (sigG) is transcribed by σ F, but also requires the activity of one of the mother-cell-specific σ-factors, σ E, for its expression. The minimal promoter required for dependence on σ E was found to stretch to just upstream of the −35 site. Analysis of mutant sigG promoters generated by site-directed mutagenesis and sigG promoters from other species suggests the presence of a binding site for a transcriptional repressor within the sigG promoter region. Replacement of the wild-type promoter with σ E-independent promoters resulted in impairment of sporulation. These data support the idea that σ E activity is required for the transcription of sigG.

This paper focuses on global gene regulation by FlhD/FlhC in enteric bacteria. Even though Yersinia enterocolitica FlhD/FlhC can complement an Escherichia coli flhDC mutant for motility, it is not known if the Y. enterocolitica FlhD/FlhC complex has an effect on metabolism similar to E. coli. To study metabolic gene regulation, a partial Yersinia enterocolitica 8081c microarray was constructed and the expression patterns of wild-type cells were compared to an flhDC mutant strain at 25 and 37 °C. The overlap between the E. coli and Y. enterocolitica FlhD/FlhC regulated genes was 25 %. Genes that were regulated at least fivefold by FlhD/FlhC in Y. enterocolitica are genes encoding urocanate hydratase (hutU), imidazolone propionase (hutI), carbamoylphosphate synthetase (carAB) and aspartate carbamoyltransferase (pyrBI). These enzymes are part of a pathway that is involved in the degradation of l-histidine to l-glutamate and eventually leads into purine/pyrimidine biosynthesis via carbamoylphosphate and carbamoylaspartate. A number of other genes were regulated at a lower rate. In two additional experiments, the expression of wild-type cells grown at 4 or 25 °C was compared to the same strain grown at 37 °C. The expression of the flagella master operon flhD was not affected by temperature, whereas the flagella-specific sigma factor fliA was highly expressed at 25 °C and reduced at 4 and 37 °C. Several other flagella genes, all of which are under the control of FliA, exhibited a similar temperature profile. These data are consistent with the hypothesis that temperature regulation of flagella genes might be mediated by the flagella-specific sigma factor FliA and not the flagella master regulator FlhD/FlhC.

Analysis of the genome sequence of the polyhydroxyalkanoate- (PHA) accumulating bacterium Ralstonia eutropha strain H16 revealed three homologues (PhaP2, PhaP3 and PhaP4) of the phasin protein PhaP1. PhaP1 is known to constitute the major component of the layer at the surface of poly(3-hydroxybutyrate), poly(3HB), granules. PhaP2, PhaP3 and PhaP4 exhibited 42, 49 and 45 % identity or 61, 62 and 63 % similarity to PhaP1, respectively. The calculated molecular masses of PhaP1, PhaP2, PhaP3 and PhaP4 were 20·0, 20·2, 19·6 and 20·2 kDa, respectively. RT-PCR analysis showed that phaP2, phaP3 and phaP4 were transcribed under conditions permissive for accumulation of poly(3HB). 2D PAGE of the poly(3HB) granule proteome and analysis of the detected proteins by MALDI-TOF clearly demonstrated that PhaP1, PhaP3 and PhaP4 are bound to the poly(3HB) granules in the cells. PhaP3 was expressed at a significantly higher level in PhaP1-negative mutants. Occurrence of an unknown protein with an N-terminal amino-acid sequence identical to that of PhaP2 in crude cellular extracts of R. eutropha had previously been shown by others. Although PhaP2 could not be localized in vivo on poly(3HB) granules, in vitro experiments clearly demonstrated binding of PhaP2 to these granules. Further analysis of complete or partial genomes of other poly(3HB)-accumulating bacteria revealed the existence of multiple phasin homologues in Ralstonia solanacearum, Burkholderia fungorum and Azotobacter vinelandii. These new and unexpected findings should affect our current models of PHA-granule structure and may also have a considerable impact on the establishment of heterologous production systems for PHAs.

A codon-profile strategy was used to predict gene expression levels in Streptococcus pneumoniae. Predicted highly expressed (PHE) genes included those encoding glycolytic and fermentative enzymes, sugar-conversion systems and carbohydrate-transporters. Additionally, some genes required for infection that are involved in oxidative metabolism and hydrogen peroxide production were PHE. Low expression values were predicted for genes encoding specific regulatory proteins like two-component systems and competence genes. Correspondence analysis localized 484 ORFs which shared a distinctive codon profile in the right horn. These genes had a mean G+C content (33·4 %) that was lower than the bulk of the genome coding sequences (39·7 %), suggesting that many of them were acquired by horizontal transfer. Half of these genes (242) were pseudogenes, ORFs shorter than 80 codons or without assigned function. The remaining genes included several virulence factors, such as capsular genes, iga, lytB, nanB, pspA, choline-binding proteins, and functions related to DNA acquisition, such as restriction-modification systems and comDE. In order to compare predicted translation rate with the relative amounts of mRNA for each gene, the codon adaptation index (CAI) values were compared with microarray fluorescence intensity values following hybridization of labelled RNA from laboratory-grown cultures. High mRNA amounts were observed in 32·5 % of PHE genes and in 64 % of the 25 genes with the highest CAI values. However, high relative amounts of RNA were also detected in 10·4 % of non-PHE genes, such as those encoding fatty acid metabolism enzymes and proteases, suggesting that their expression might also be regulated at the level of transcription or mRNA stability under the conditions tested. The effects of codon bias and mRNA amount on different gene groups in S. pneumoniae are discussed.

The extremely thermophilic bacterium Thermus thermophilus HB27 synthesizes lysine through α-aminoadipate (AAA). In this study, a T. thermophilus gene encoding the enzyme that catalyses transamination of AAA was cloned as a mammalian kynurenine/AAA aminotransferase (Kat2) gene homologue. A T. thermophilus mutant with disruption of the Kat2 homologue required a longer lag phase for growth and showed slower growth in minimal medium. Furthermore, addition of AAA or lysine shortened the lag phase and improved the growth rate. The Kat2 homologue was therefore termed lysN. LysN recognizes not only 2-oxoadipate, an intermediate of lysine biosynthesis, but also 2-oxoisocaproate, 2-oxoisovalerate and 2-oxo-3-methylvalerate, intermediates of leucine, valine and isoleucine biosyntheses, respectively, along with oxaloacetate, a compound in the TCA cycle, as an amino acceptor. These results suggest multiple roles of LysN in several cellular metabolic pathways including lysine and branched-chain amino acid biosyntheses.

The FixLJ two-component system of Sinorhizobium meliloti is a global regulator, turning on nitrogen-fixation genes in microaerobiosis. Up to now, nifA and fixK were the only genes known to be directly regulated by FixJ. We used a genomic SELEX approach in order to isolate new FixJ targets in the genome. This led to the identification of 22 FixJ binding sites, including the known sites in the fixK1 and fixK2 promoters. FixJ binding sites are unevenly distributed among the three replicons constituting the S. meliloti genome: a majority are carried either by pSymA or by a short chromosomal region of non-chromosomal origin. Thus FixJ binding sites appear to be preferentially associated with the pSymA replicon, which carries the fixJ gene. Functional analysis of FixJ targets led to the discovery of two new FixJ-regulated genes, smc03253 and proB2. This FixJ-dependent regulation appears to be mediated by a duplication of the whole fixK promoter region, including the beginning of the fixK gene. Similar duplications were previously reported for the nifH promoter. By systematic comparison of all promoter regions we found 17 such duplications throughout the genome, indicating that promoter duplication is a common mechanism for the evolution of regulatory pathways in S. meliloti.

Enteropathogenic and enterohaemorrhagic Escherichia coli (EPEC and EHEC, respectively) genomes contain a pathogenicity island, termed the locus of enterocyte effacement (LEE), which encodes genes involved in the formation of attaching and effacing lesions on epithelial cells. To elucidate the regulatory mechanism of the LEE genes in EHEC, an EHEC O157 genomic library was screened for clones which modulated expression of the LEE genes. From more than 5000 clones, a DNA fragment was obtained containing a perC homologue as a positive regulator for the LEE genes. In EPEC, perC is known to be part of the per operon, along with perA and perB, located on the EPEC adherence factor plasmid, which is not found in EHEC. However, the complete genome sequence of EHEC O157 Sakai strain reveals that there are five perC-like sequences, but no perA and perB, on the chromosome. These five perC homologues were characterized, and it was found that three of the homologues (renamed perC homologue pchA, pchB and pchC) encoded 104 aa proteins, and when expressed on a multicopy plasmid enhanced the expression of LEE genes. In contrast, perC homologues encoding proteins of 89 and 90 aa, renamed pchD and pchE, respectively, had no significant effect. Deletion mutants of the pch genes were constructed, and the effect on the expression of LEE-encoded type III effector proteins, such as EspA, B and D, and adhesion phenotype to HEp-2 cells was examined. Deletion of pchA or pchB, but not pchC, decreased the expression of Esp proteins and adhesion to HEp-2 cells. Such effects were more apparent with mutants carrying double deletions of pchA/pchB or pchA/pchC, suggesting that pchA/B/C are all necessary for full expression of the LEE genes and adhesion to HEp-2 cells. Further study demonstrated that the positive effect of pchA/B/C was caused by enhanced transcription of the LEE-encoded regulatory gene, ler. Introduction of a multicopy plasmid carrying each pchA/B/C gene significantly induced microcolony formation by EHEC O157 on HEp-2 cells. These results suggest that the pchABC genes are necessary for full virulence of EHEC O157.

Fusobacterium nucleatum can bind to saliva-coated tooth surfaces. However, the nature of the domains of salivary protein that interact with F. nucleatum remains unclear. The ability of individual proteins in human submandibular-sublingual saliva (HSMSL) to bind F. nucleatum cells was examined by dot blot assay; statherin displayed the strongest binding activity. Statherin binding sites were determined based on binding of 125I-labelled F. nucleatum to statherin-coated hydroxyapatite (sHAP) beads via inhibition assays using synthetic analogous peptide fragments of whole statherin. Analogous peptides corresponding to residues 19–26 and 32–39 of statherin inhibited binding by 77 % and 68 %, respectively. Synthetic peptides were also prepared by serial deletions of individual residues from N- and C-termini of the peptides GPYQPVPE (aa 19–26) and QPYQPQYQ (aa 32–39). The inhibitory effects of peptides YQPVPE (aa 21–26) and PYQPQYQ (aa 33–39) were very similar to those of GPYQPVPE and QPYQPQYQ, respectively. However, additional deletion of residues resulted in significant reduction of the inhibitory effect. Alanine-scan analysis of YQPVPE revealed that all tested peptides retained inhibitory activity; only YAPVPE exhibited significantly decreased inhibitory activity. These findings suggest that YQPVPE and PYQPQYQ may represent the minimal active segments of statherin for binding to F. nucleatum; moreover, Gln may be a key amino acid in the active segment.

Paenibacillus larvae subsp. larvae (P. l. larvae) is the aetiological agent of American foulbrood (AFB), the most virulent bacterial disease of honey bee brood worldwide. In many countries AFB is a notifiable disease since it is highly contagious, in most cases incurable and able to kill affected colonies. Genotyping of field isolates of P. l. larvae revealed at least four genotypes (AB, Ab, ab and α B) present in Germany which are genotypically different from the reference strain DSM 7030. Therefore, based on these data, five different genotypes of P. l. larvae are now identified with genotype AB standing out with a characteristic brown-orange and circled two-coloured colony morphology. Analysing the metabolic profiles of three German genotypes (AB, Ab and ab) as well as of the reference strain using the Biolog system, a characteristic biochemical fingerprint could be obtained for each strain. Cluster analysis showed that while genotypes Ab, ab and the reference strain DSM 7030 are rather similar, genotype AB is clearly different from the others. Analysis of all isolates for plasmid DNA revealed two different plasmids present only in isolates belonging to genotype AB. Therefore, genotype AB is remarkable in all aspects analysed so far. Future analysis will show whether or not these differences will expand to differences in virulence.

Actinobacillus pleuropneumoniae serotype 1 adhered to immobilized swine-lung collagen. Bacteria bound to collagen type I, III, IV and V. At 5 min incubation, 30 % of bacteria adhered to collagen, reaching saturation in around 90 min. Treatment of bacteria with divalent-metal chelators diminished their attachment to collagen, and Ca2+ but not Mg2+ increased it, suggesting Ca2+ dependence for adherence. Proteolytic enzymes drastically reduced bacterial adherence to collagen, showing that binding involved bacterial surface proteins. Porcine fibrinogen, haemoglobin and gelatin partially reduced collagen adhesion. A 60 kDa outer-membrane protein of A. pleuropneumoniae recognized the swine collagens by overlay. This membrane protein was apparently involved in adhesion to collagen and fibrinogen, but not to fibronectin and laminin. Antibodies against the 60 kDa protein inhibited the adhesion to collagen by 70 %, whereas pig convalescent-phase antibodies inhibited it by only 40 %. Serotypes 1 and 7 were the most adherent to pig collagen (taken as 100 %); serotypes 6 and 11 were the lowest (∼50 %), and neither showed the 60 kDa adhesin to biotinylated collagens. By negative staining, cells were observed initially to associate with collagen fibres in a polar manner, and the adhesin was detected on the bacterial surface. The results suggest that swine-lung collagen is an important target for A. pleuropneumoniae colonization and spreading, and that the attachment to this protein could play a relevant role in pathogenesis.

Treponema denticola is a major aetiological organism implicated in periodontal disease. The interaction of T. denticola with other oral bacteria, in particular Porphyromonas gingivalis, in biofilm formation is thought to be an important step in the onset of periodontal disease. The interaction between T. denticola and P. gingivalis has been examined using a panel of T. denticola mutants and their effects on mixed biofilm formation tested in a static biofilm model. T. denticola ATCC 35405 did not form detectable biofilms on various inert surfaces. However, the spirochaete was demonstrated to form a biofilm with preattached P. gingivalis 381. T. denticola cfpA, which lacks the cytoplasmic filament, was unable to produce a mixed biofilm with P. gingivalis. A T. denticola flgE mutant which lacks the flagella hook protein and is therefore non-motile displayed a reduced, but readily detectable, ability to form a mixed biofilm as did the T. denticola mutant which does not possess the major outer sheath protein (Msp). The T. denticola lrrA mutant was only moderately defective in forming mixed biofilms with P. gingivalis. However, the T. denticola methyl-accepting chemotaxis protein (DmcA) did not appear to play a major role in mixed biofilm formation. In contrast, T. denticola lacking the PrtP protein for prolyl-phenylalanine-specific protease, showed an increased ability to form mixed biofilms and a prolonged viability in the biofilm.

Streptococcus pneumoniae is a human pathogen that is naturally transformable. In this study a major component of the homologous recombination pathway, the RexAB exonuclease/helicase, was characterized. rexA and rexB insertional mutants were constructed using mariner mutagenesis and found to have identical phenotypes. Both rexAB mutants displayed poor cell viability, reduced double-strand exonuclease activity, UV sensitivity and a reduced level of gene conversion compared to the wild-type strain. No effect was observed on plasmid and chromosomal transformation efficiencies. These results indicate that in S. pneumoniae, RexAB is required for DNA repair, but not for chromosomal transformation and plasmid establishment.